effect of continuous light on daily levels of plasma ...ruoff/salmon2.pdf · effect of continuous...

EFFECT OF CONTINUOUS LIGHT ON DAILY LEVELS OF PLASMAMELATONIN AND CORTISOL AND EXPRESSION OF CLOCKGENES IN PINEAL GLAND, BRAIN, AND LIVER IN ATLANTICSALMON POSTSMOLTS

Tien-sheng Huang,1 Peter Ruoff,2 and Per G. Fjelldal3

1National Institute of Nutrition and Seafood Research (NIFES), Nordnes, Bergen,Norway2Centre for Organelle Research, University of Stavanger, Stavanger, Norway3Institute of Marine Research (IMR), Matredal, Norway

Continuous light is a common practice in salmon farming, where it is used toenhance growth, induce smoltification, and regulate puberty. However, knowledgeabout how different tissues receive information about daylength is limited. The aimof the present study was to evaluate the daily expression of clock (Per1-like, Cry2, andClock), the nuclear transcription factor (peroxisome proliferator-activated receptor,PPAR; CCAAT/enhancer binding protein, C/EBP), and the endoplasmic reticulum(ER) stress (protein disulfide isomerase associated 3, PDIA3) genes in the pinealgland, brain, and liver of Atlantic salmon postsmolts reared under 12-h light:12-hdark (LD) regimes or under continuous light (LL) for 6 wks following transfer to sea-water. All measured clock mRNAs displayed daily variations in one or more organsunder LD, as well as plasma levels of melatonin. Similar variations were noted in theliver c/ebpα, pineal c/ebpδ, and pdia3 mRNAs. Under LL, the clock and nuclear tran-scription factor mRNAs did not show any daily variation in the studied organs, withthe exception of pineal pdia3. Furthermore, LL had the opposite effect on the levelsof melatonin and cortisol, as observed by the increase in pineal Clock, Per2, pparα,and c/ebpα and c/ebpδ mRNAs and decrease in liver Clock, Per2, and pparα mRNAscompared to those under LD. The present findings show that the expression of clockgenes is affected by the light across organs and that there is a relation between PPAR,C/EBP, and clock mRNAs; however, the functional role of the individual nucleartranscription factors related to this observation remains to be established in the pinealgland and liver. (Author correspondence: [email protected])

Keywords Atlantic salmon; Clock gene; Cortisol; Light; Melatonin; Nucleartranscription factor

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Address correspondence to Dr. Tien-sheng Huang, National Institute of Nutrition and SeafoodResearch (NIFES), P. O. Box 2029 Nordnes, N-5817 Bergen, Norway. Tel: (47) 98859588; Fax: (47)55905299; E-mail: [email protected]

Submitted April 7, 2010, Returned for revision May 7, 2010, Accepted August 11, 2010

Chronobiology International, 27(9–10): 1715–1734, (2010)Copyright © 2010 Informa Healthcare USA, Inc.ISSN 0742-0528 print/1525-6073 onlineDOI: 10.3109/07420528.2010.521272

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INTRODUCTION

Molecular clocks control the daily variations of wakefulness, stresshormones, lipid metabolism, immune function, and cell division, amongother functions and processes (Duez & Staels, 2008). Genetic studies haveidentified components of the circadian pacemaker, including negativeregulators, period (PER) and cryptochrome (CRY), as well as positive reg-ulators, CLOCK and BMAL1, which are conserved in mammals wherethey constitute the core of the circadian clock described as transcrip-tional-translational feedback loops (Duguay & Cermakian, 2009; Gauger& Sancar, 2005). These feedback loops control the central and peripheralclocks that generate and maintain the functions and properties of varioustissues and cell types during constant environmental conditions(Balsalobre, 2002; Ralph et al., 1990). Circadian clocks can be induced insome cultured cell lines and stem cells in vitro by different stimuli(Balsalobre et al., 1998; Huang et al., 2009a; Mendez-Ferrer et al., 2008).In contrast to the mammalian biological clock located in the suprachias-matic nucleus (SCN) of the brain, to date the location of biological clockin fish is not known. Although the zebrafish is a useful model to study theeffects of environmental stimuli, including light and temperature on cir-cadian clock mechanisms (Hirayama et al., 2005; Lahiri et al., 2005),related studies are still lacking in other teleosts. Recently, Clock and Per1has been mapped to a quantitative trait locus to act as potential candi-dates for the timing of spawning in rainbow trout (Leder et al., 2006),and daily variations in clock gene expression have been shown in Atlanticsalmon parr brains (Davie et al., 2009) and pineal glands (Huang et al.,2010b). These studies suggest the existence of a clock gene system thatcouples inputs from the environment to complex biological functions andcontrols the timing of lifecycle transitions in salmonids.

In seasonal mammals, robust annual cycles of adiposity, food intake,and energy metabolism are driven by daylength, which generates adiurnal melatonin profile and acts on neuroendocrine pathways(Rousseau et al., 2003). Salmonids also exhibit behavioral and physiologi-cal rhythms, of which the generation of the biological timing system isthought to control their daily and annual functions in relation to develop-ment, locomotor activity, sedation, skin pigmentation, oxygen consump-tion, and thermoregulation (Falcon et al., 2010). Seasonal changes inphotoperiod are the most predictable environmental cues to modulatedaily and annual rhythms. Daylength controls the release of pineal mela-tonin in the blood, with low and high levels during the light and darkphases, respectively (Falcon, 1999). In Atlantic salmon, photoperiod influ-ences behavior (Oppedal et al., 2001); appetite (Nordgarden et al.,2003a); muscle lipid composition (Nordgarden et al., 2003b); develop-ment and growth of the axial muscle (Johnston et al., 2003), vertebral

T.-s. Huang et al.1716

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column (Fjelldal et al., 2005), and gonads (Taranger et al., 1998); as wellas plasma levels of melatonin (Porter et al., 1999), cortisol (Thorpe et al.,1987), growth hormone (GH), insulin-like growth, factor 1 (IGF-I)(Nordgarden et al., 2005), and sex steroids (Taranger et al., 1998).Cortisol and GH act together during smoltification and seawater adaptionin regulating gill sodium-potassium adenosine triphosphatase (Na+,K+-ATPase) activity (McCormick, 2001). However, light-induced effects onthe temporal relationship between physiological clocks and plasma levelsof melatonin and cortisol are not clear in Atlantic salmon postsmolts.

CCAAT/enhancer binding protein (C/EBP) in the mollusc eye(Hattar et al., 2002), C/EBPδ in the hamster pineal gland (Marondeet al., 2007), and peroxisome proliferator-activated receptor (PPARα) inthe rat liver (Lemberger et al., 1996) have been identified as clock-controlled genes with diurnal expression. In addition, PPARα is alsoinvolved in the circadian regulation of lipid metabolism (Oishi et al.,2005). In Atlantic salmon, PPAR and C/EBP display important roles inregulating lipid metabolism in hepatocytes, adipocytes, and myocytes(Huang et al., 2010a; Kleveland et al., 2006; Vegusdal et al., 2004), andstress-induced pdia3 mRNA expression was found in the liver of smoltssuffering from hypoxia (Huang et al., 2009b). Less is known about theassociation of clock and nuclear transcription factor mRNAs in teleost.

The clock gene system has previously been shown in Atlantic salmonparr and smolts (Davie et al., 2009; Huang et al., 2010b), whereas theexpression of clock and clock-controlled genes and their response to lighthas not been reported in neural and peripheral tissues in postsmolts. Inorder to better understand the daily expression of clock and nuclear tran-scription factor mRNAs in Atlantic salmon in seawater, we described theplasma levels of melatonin and cortisol, in addition to the expression ofclock (Per1-like, Per2, Cry2, and Clock), nuclear transcription factor ( pparand c/ebp), and endoplasmic reticulum (ER) stress ( pdia3) mRNAs in thepineal gland, brain, and liver of postsmolts reared under 24 h of continu-ous light (LL) or a 12-h light:12-h dark (LD) cycle.

MATERIALS AND METHODS

Fish Stock

The underyearling Atlantic salmon (Salmo salar L.) smolts used in thepresent experiment were produced at the Institute of Marine Research(IMR), Matre Research Station, western Norway. The fish were from theAquagen strain, and smoltification was induced by an artificial photo-period commonly used in commercial salmon farming. The fish hadbeen reared under LD for 6 wks, followed by LL for 6 wks in freshwateruntil December 14, 2007, when they were transferred to experimental

Clock Gene Expression in Postsmolts 1717

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seawater tanks. On December 14, the smolts had a low condition factor,lacked parr marks, and were silvery, and the mean gill Na+,K+-ATPaseactivity was 9.2 mmol adenosine diphosphate (ADP)/mg protein/h (minvalue 6.9, max value 12.5), which confirmed complete smoltification.

Experimental Design

On December 14, 2007, 300 underyearling smolts (mean weight196 g) were randomly allocated to six square, white, covered seawatertanks (1 × 1 × 0.43 m), with 50 fish/tank. For the following 6 wks (fromDecember 14, 2007 to January 25, 2008), triplicate groups in separatetanks were employed with LD or LL. For illumination, two 18-W fluor-escent daylight tubes (OSRAM L 18W/840 LUMILUX; OSRAM,Ausburg, Germany) were used to produce 960 lux underwater in thecenter of the tank. The light of the LD tanks was switched on at 08:05 hand off at 20:05 h. Both the LD and LL groups were fed by disc feedersthat continuously release pellets into the water during the lighted periodfor the LD group. The feed used was a commercial salmon feed (NutraOlympic; Skretting, Averøy, Norway), and the groups were fed by auto-matic feeders (ARVO-TEC T Drum 2000; Arvotec, Huutokoski, Finland).The fish were starved 24 h prior to sampling. The light and feeding werecontrolled automatically by a PC-operated system (Normatic, Norfjordeid,Norway). The temperature was stable at 8.9°C, and the oxygen saturationof the outlet was kept >80%.

Sampling of Atlantic Salmon Tissues for Expression Analysis

The samplings were performed on January 24–25, 2008, withsampling performed every 4 h; the first sampling started at 12:00 h onday 1 and the last started at 12:00 h on day 2 (day 1: 12:00, 16:00, 20:00,24:00 h, and day 2: 04:00, 08:00, and 12:00 h). At each sampling, 3 fish/tank were anesthetized with benzokain (40 mg L−1) (Benzoak® Vet;A.C.D., Braine-L’alleud, Belgium) and measured for fork length andbody weight. Blood samples were then taken from the caudal vessel usinga heparinized tuberculin syringe fitted with a 23-gauge needle. Plasmawas separated by centrifugation and stored at −80°C until analysis. Thenight sampling of individual fish of the LD tanks was performed under adim-red light. The fish were then decapitated with a scalpel, and thetissues were dissected from the carcass and immediately frozen in liquidnitrogen. During the sampling, all fish were anesthetized at the sametime, and then 1 fish/tank was sequentially measured and dissected toomit systematic errors caused by time-related differences in geneexpression between the tanks and light groups within the subsamplings.The experimental protocol and procedures were performed in


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accordance with both international ethical standards (Portaluppi et al.,2008) and the National Animal Research Authority in Norway.

Levels of Plasma Melatonin and Cortisol

Melatonin levels were determined by radioimmunoassay (RIA) kit, inwhich the plasma sample was incubated with assay buffer, 125I-melatonin,and rabbit anti-melatonin antiserum, as described in the manufacturer’sinstructions (IBL, Hamburg, Germany). The cortisol concentration wasmeasured by RIA kit (DiaSorin, Minnesota, USA), in which 10 μL plasmasamples were applied in 1 mL tracer-buffer before incubation with125I-cortisol and rabbit anti-corticosterone antibody followed by thestandard protocol (Stefansson et al., 1989).

Isolation of RNA and qRT-PCR Analysis

RNA isolation and DNase treatment of the tissue samples were per-formed using a standard RNA extraction method (Invitrogen, Paisley,UK) and DNA-free™ DNase following the manufacturer’s protocol. Thetotal RNA, 500 ng/sample, was reverse transcribed using TaqMan RTreagents (ABI, Foster City, CA, USA) according to the manufacturer’sinstructions. A standard curve was obtained from a two-fold serial dilutionof total RNA from the control fish, with six RNA concentrations coveringbetween 31.25 ng and 1000 ng. The quantitative reverse transcriptase–-polymerase chain reaction (qRT-PCR) primer sequences (Table 1) weredesigned from expressed sequence tag (EST) sequences of Per2, Cry2,and Clock, obtained from the Salmon Genome Project (SGP) database,www.salmongenome.no, as described by Davie et al. (2009). In order toidentify salmon Per ortholog from the EST sequence, PCR primers, 5′-CCCAGTGACCTACTGGACCTG-3′ and 5′-TGGTGCTCTCTGGTAAGG-3′, were designed to amplify a partial cDNA fragment (576 bp) fromAtlantic salmon liver mRNA by PCR reaction. We refer to this gene as thePer1-like gene. The PCR primers for PPAR and C/EBP genes weredesigned from published sequences of Atlantic salmon (Huang et al.,2010a). The amplified PCR products of all three clock genes weresequenced to ensure correct mRNA sequences for quantification. Theprimers shown in Table 1 were used for SYBR Green assays (RocheDiagnostics, Germany) in a total volume of 20 μL containing 1 μL cDNA,10 μL SYBR Green master, 0.2 μL (50 μM) of each primer, and 8.6 μLH2O. Reactions were run on a Roche Lightercycler 480 (RocheDiagnostics) with 96 wells. The cycling conditions were composed of pre-incubation at 95°C for 5 min, followed by 45 cycles of amplification. Eachcycle included 10 s of denaturation at 95°C, 20 s of primer annealing at60°C, and 30 s of extension/synthesis at 72°C. The gene expression


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TABLE 1 Primer sequences used in qRT-PCR of the reference and clock genes studied

Gene Accession number Forward primer Reverse primer

pdia3 EJ457023 5′-CCAACGCCATGATCAAGAA-3′ 5′-CAGCAGGTCCTTGTCCTTCA-3′

c/ebpα EU668995 5′-AGACCTCGGCGAGATTTGT-3′ 5′-TGTGGAATAGATCAGCCAGGAA-3′

c/ebpδ EU668997 5′-TTGGGCGGTGGAGCCTAT-3′ 5′-TTTCCTCGCCCGTGTCAT-3′

pparα DQ294237 5′-TCCTGGTGGCCTACGGATC-3′ 5′-CGTTGAATTTCATGGCGAACT-3′

Per1-like DW576689 5′-CTGTCTCCTTGGGCACTGTGT-3′ 5′-GAGTCGATGCTGCCAAAGTACTT-3′

Clock CA038738 5′-GTTAGACGGCTTCTTCCTAGCAAT-3′ 5′-CCACCAGGTCAGAAGGAAGATG-3′

Cry2 DY730105 5′-CCACAACATGGCAGGTGAATT-3′ 5′-CAGACATCCAAAGCGGAGGTA-3′

Per2 FM877775 5′-GCTCCCAGAATTCCTAGTGACAAG-3′ 5′-GAACAGCCCTCTCGTCCACATC-3′

β-Actin BG933897 5′-CCAAAGCCAACAGGGAGAAG-3′ 5′-AGGGACAACACTGCCTGGAT-3′

EF1AB AF321836 5′-TGCCCCTCCAGGATGTCTAC-3′ 5′-CACGGCCCACAGGTACTG-3′

ARP AY255630 5′-GAAAATCATCCAATTGCTGGATG-3′ 5′-CTTCCCACGCAAGGACAGA-3′

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stability (M) was calculated with geNorm, and the three reference genes(ARP, β-actin, and EF1AB) revealed a low M value. The geNorm tool wasused to determine a normalizing factor from the three reference genes(acidic ribosomal phosphoprotein [ARP], β-actin, and elongation factor 1AB [EF1AB]) to postsmolts (LD and LL), and it was also used to calculatethe normalized expression for the target genes (Vandesompele et al.,2002).

Statistical Analysis

Significant differences in both length and weight were tested by two-way nested analyses of variance (ANOVAs), with the tank as the randomfactor nested in the light group, and those in the plasma levels of melato-nin and cortisol and in the mRNA expression of each clock, nuclear tran-scription factor, and pdia3 genes were evaluated by one-way ANOVA(Statistica; Statsoft, Tulsa, USA). If the ANOVA test showed statistical sig-nificance ( p< .05), a Newman-Keuls test was applied to determine allpairwise comparisons. Because ANOVA cannot assess rhythmicity, thecosinor method was used to test for circadian rhythmicity using normaliz-ing data for each variable in three individual analyses (the average of the 3fish/tank, n= 3 each timepoint) and in one cosinor test using the meansfor each timepoint (means of total fish from all the tanks). These analyseswere performed by the cosinor periodogram (www.circadian.org/softwar.html), with significance of the periodicity determined by the zero-ampli-tude test of the cosinor procedure. The rhythm characteristics estimatedby this method include the mesor (middle value of the fitted cosine repre-senting a rhythm-adjusted mean), amplitude (half the difference betweenthe minimum and maximum of the fitted cosine function), and acrophase(the time of the peak value expressed in h and min from local 00:00 h)(Table 2).

RESULTS

Survival, Somatic Growth, and Plasma Levels of Melatonin andCortisol

There was no mortality in either of the groups during the 6-wkexperimental period, and no significant differences in fork length andbody weight between the fish under LD (27.9± 0.3 cm, 249± 7.3 g) andLL (27.5± 0.2 cm, 227± 6.1 g) at the end of the experiment. Under LDconditions, plasma melatonin levels were typically low during the lightphase and gradually increased during the dark phase (cosinor, p< .01)(Figure 1) in contrast with no significant difference at any timepoint inmelatonin levels under LL (ANOVA, p = .55). There was no significant


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TABLE 2 Summary of clock gene expression and plasma melatonin from cosinor analysis inpostsmolts

Gene Tissue Mesor± SD Amplitude± SD Acrophase Cosinor p

12-h light:12-h dark (LD)Per1-like Liver 0.58± 0.02 0.19± 0.01 23:21± 0.46 <.01

Brain 0.29± 0.01 0.16± 0.01 06:46± 0.02 <.05Pineal 0.36± 0.01 0.20± 0.03 03:54± 0.25 <.01

Cry2 Brain 0.44± 0.01 0.24± 0.01 05:44± 0.22 <.05Pineal 0.25± 0.01 0.15± 0.01 23:48± 0.23 <.01

Clock Liver 0.76± 0.04 0.21± 0.04 04:00± 1.29 <.05Brain 0.38± 0.02 0.25± 0.03 08:52± 0.10 <.05

Per2 Brain 0.43± 0.02 0.35± 0.03 08:34± 0.22 <.05Pineal 0.38± 0.01 0.15± 0.02 23:53± 0.75 <.05

c/ebpα Liver 0.51± 0.03 0.14± 0.03 23:58± 0.50 <.05c/ebpδ Pineal 0.51± 0.17 0.20± 0.04 04:14± 1.11 <.05pdia3 Pineal 0.30± 0.01 0.10± 0.01 21:32± 0.90 <.01Melatonin Plasma 1033± 94 1199± 129 04:28± 0.17 <.01

Continuous light (LL)pdia3 Pineal 0.58± 0.02 0.25± 0.02 20:21± 0.52 <.05

FIGURE 1 Daily plasma levels of melatonin and cortisol in Atlantic salmon postsmolts. Plasma levelsof melatonin and cortisol were measured from postsmolts reared under 12 h of light:12 of h dark(LD) or 24 h of continuous light (LL) for 6 wks following transfer to seawater. Circadian rhythmswere determined by three separate cosinor analyses and the statistical significance is reported in thetext. Each value is the mean± SEM (n= 3; 3 fish/tank). LD, 12-h light:12-h dark; LL, continuouslight. N= 9; 3/tank.

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daily variation in plasma cortisol in fish reared under LL (ANOVA, p =.86) or LD (ANOVA, p < .01; cosinor, p = .97), although the level wastwo-fold higher under LL compared to LD (ANOVA, p < .001)(Figure 1).

Variations of Daily Clock Gene Expression in Salmon Postsmolts

Under LD, there was significant daily variation of Per1-like in thepineal gland, brain, and liver; of Cry2 and Per2 in the pineal gland andbrain, and of Clock in the brain and liver (Figure 2). The mesor andamplitude were similar in the pineal gland and brain for Per2, Per1-like,and Cry2, and their acrophases, respectively, occurred in the pineal gland2 h before (23:53 h), 2 h after (03:54 h), and 2 h before (23:48 h) themiddle of the dark period (02:00 h), respectively, whereas their

FIGURE 2 Daily expression of clock gene mRNAs in the pineal gland, liver, and brain of Atlanticsalmon postsmolts. Expression of clock gene mRNAs in the pineal gland, brain, and liver harvestedfrom postsmolts reared under 12 h of light:12 h of dark (LD) or 24 h of continuous light (LL) for 6wks following transfer to seawater and normalized to three endogenous control genes, ARP, β-actin,and EF1AB. Circadian rhythms were determined in three separate cosinor analyses, and the statisticalsignificance is shown in Table 2. Each value is the mean± SEM (n= 3; 3 fish/tank). LD, 12-hlight:12-h dark; LL, continuous light; n= 9, 3/tank.


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acrophases in the brain for Per1-like and Cry2 occurred late in the darkperiod at 06:46 and 05:44 h, respectively (Table 2). In contrast, the acro-phase of brain Per2 and Clock was early in the light phase, at 08:34 and08:52 h, respectively (Table 2). The acrophases of liver Per1-like and Clockoccurred 2.5 h before (23:21 h) and 2 h (04:00 h) after the middle of thedark period, respectively (Table 2). Of the studied genes and organs, thepineal and liver Per1-like and the pineal Cry2 demonstrated the most pro-nounced oscillation ( p < .01; Table 2), and only pineal Per1-like demon-strated a strong response after the shift between the light and dark spans(marked increase between 20:00 and 24:00 h; Figure 2).

Under LL, none of the clock RNAs showed significant daily variationsin any of the analyzed tissues, but the overall levels of the Clock and Per2increased in the pineal gland and decreased in the liver of LL-reared fish(Figure 2).

Rhythmic Expression of Nuclear Transcription Factor and ERStress-Related Genes

The mRNA expression of c/ebpα and c/ebpδ displayed significant dailyvariations in the liver and pineal gland, respectively, with acrophases 2 hbefore (23:58 h) and 2.2 h (04:14 h) after the middle of the dark period(Figure 3, Table 2). The pparα expression did not show significantrhythms in either the pineal gland or liver. There were no rhythms ofthese mRNAs in fish under LL, but mRNA levels of the liver pparα wererepressed by LL and those of the pineal pparα, c/ebpα, and c/ebpδ wereup-regulated by LL when compared to those under LD. The pdia3expression showed significant daily variations in the pineal gland of fishreared under both LD and LL, but their respective acrophases weredifferent under the two light conditions (Figure 3, Table 2).

DISCUSSION

The present study is the first to demonstrate the effect of light ondaily variations in the expression of genes encoding for clock proteins,nuclear transcription factors, and ER stress protein in seawater postsmoltsof Atlantic salmon. We observed daily variations of plasma melatoninlevels under LD, which were elevated in the dark phase and suppressedin the light phase. This nocturnal rise in plasma melatonin disappearedunder LL. These results are in accordance with reports on the diurnalrhythms of plasma melatonin levels in salmonids (Iigo et al., 1997;Randall et al., 1995). Although significant daily variations of cortisollevels were not seen in postsmolts (LD), lower levels occurred during thetransition between the day and night spans, which was in contrast withthe nocturnal increase in plasma cortisol reported in smolts under


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natural winter photoperiods (Ebbesson et al., 2008). Some studies havereported daily variations of cortisol with different acrophases in fish, suchas rainbow trout (Polakof et al., 2007), goldfish (Singley & Chavin, 1975),carp (Kuhn et al., 1986), eel (Li & Takei, 2003), and sea bass (Cerda-Reverter et al., 1998), which may be due to different light regimes andspecies. LL induced overall higher plasma cortisol levels compared toLD. In rainbow trout, glucocorticoid receptors are present in the pineal

FIGURE 3 Daily expression of nuclear transcription factor and pdia3 mRNAs in the pineal glandand liver of Atlantic salmon postsmolts. Expression of PPAR, C/EBP, and pdia3 mRNAs in the pinealgland and liver harvested from postsmolts reared under 12 h of light:12 h of dark (LD) or 24 h ofcontinuous light (LL) for 6 wks following transfer to seawater and normalized to three endogenouscontrol genes, ARP, β-actin, and EF1AB. Circadian rhythms were determined in three separatecosinor analyses and the statistical significance is shown in Table 2. Each value is the mean± SEM(n= 3; 3 fish/tank). LD, 12-h light:12-h dark; LL, continuous light; n= 9, 3/tank.


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gland and melatonin production is inhibited by administration of the syn-thetic glucocorticoid dexamethasone (Benyassi et al., 2001). Similarresults were found in mammals, in which adrenocorticotropin-stimulatedcortisol was inhibited by the administration of melatonin (Torres-Farfanet al., 2003). Moreover, maternal melatonin suppression increases cortisolproduction in the newborn rat (Torres-Farfan et al., 2004). Therefore,there may be an inverse relationship between melatonin and cortisol inAtlantic salmon under LL, which needs to be studied in more detail.Cortisol is known to be involved in the endocrine response to stress(Boujard & Leatherland, 1992). In the present study, however, it is unli-kely that the cortisol levels were a result of stress during the capture pro-cedure, since they did not reveal high levels in both LD and LL. Inteleost, it is known that cortisol increases salinity tolerance through itseffect on mineral corticoids and ion transport proteins (Kiilerich et al.,2007; McCormick et al., 2008). Thus, cortisol levels appear to be influ-enced by light, although the biological function of the constant increaseof levels induced by LL is as yet unknown in postsmolts.

Here, the rhythmic expression of the pineal and liver Per1-like peakedat midnight, which is in accordance with reports in goldfish retina Per1(Velarde et al., 2009). The peak of brain Per1-like that occurred at dawnwas similar to that of Per1b (previously referred to as Per4) in the goldenrabbitfish brain (Park et al., 2007). In zebrafish, rhythmic expression ofPer1a (previously referred to Per1), Per1b, Per2, and Per3—with differenttiming of their maximal expression—was reported in an in vitro embryosystem, in which those of Per1a, Per1b, and Per3 were activated beforelight-onset (Pando et al., 2001; Vallone et al., 2004). In mice, three Pergenes—Per1, Per2, and Per3—are rhythmically expressed with differentlight responses in the SCN, but only Per1 and Per2 have been identifiedas important elements of the feedback loops (Takumi et al., 1998a,1998b; Zylka et al., 1998). We have described the expression of Atlanticsalmon Per1-like gene in parr and smolts (Huang et al., 2010b), in whichthe putative amino acid sequence derived from a cDNA fragment of Per1-like showed greatest homology with golden rabbitfish PER1b (Park et al.,2007). Since the full sequence data were not obtained, it is necessary toobtain a large sequence in order to clarify the identity of Per1 expression.

Similar to light-dependent Per2 expression in zebrafish (Delaunayet al., 2000; Ziv et al., 2005), golden rabbitfish (Sugama et al., 2008), andgoldfish (Velarde et al., 2009), brain Per2 mRNAs oscillated under LDwith a peak of expression at 08:34 h, which corresponds to the time oflight-onset. However, the peak expression of pineal Per2 occurred in thedark phase, as described in parr brain Per2 under a short-day photo-period (SD) by Davie et al. (2009). It is likely that Per2 expression in thewhole brain does not occur in parallel with that in the pineal gland,which does not contain a functional circadian clock as in other teleosts


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(Falcon et al., 2010). We have unpublished data on pituitary clock geneexpression in smolts showing that light-dependent Per2 expression is anti-phasic to Per1-like; this suggests that other brain areas contribute tointernal circadian oscillators and that the regulation of Per2 is different insalmonids compared to other teleosts (Sugama et al., 2008; Velarde et al.,2009; Ziv et al., 2005). Although we cannot explain why Per2 is light-induced in the pineal gland but not in the liver and brain under LL inthe present study, this finding can provide important information aboutthe clock gene system in salmonids.

Pineal Cry2 peaked at midnight and brain Cry2 peaked at dawn,which is similar to the nocturnal expression of Cry2 in goldfish retina(Velarde et al., 2009), Atlantic salmon parr brain (Davie et al., 2009), andzebrafish larva (Lahiri et al., 2005). The collection of these resultssuggests that Atlantic salmon Per1-like and Cry2 mRNAs are expressedwith daily variations in the brain and peripheral tissues of postsmolts.The peak expression of brain Clock was in the beginning of the lightphase (08:52 h), and in liver Clock it was in the late night phase; in con-trast, Atlantic salmon parr Clock peaked at midnight under SD (Davieet al., 2009), whereas it peaked during the light phase in mouse SCN(Abe et al., 1999). These variations of the peak time may imply that thereis a different response of Clock expression in various organs, species, andunder different photoperiods. Given that Clock expression is not rhythmicin the pineal gland under LD, we suggest that Per1-like and Cry2expression is not dependent on the rhythmic expression of Clock in thepineal gland, although it is unknown whether this organ acquires specificregulation of clock gene expression in postsmolts. The mechanisms regu-lating gene expression in the rat pineal gland are complex, because genetranscription in this organ is controlled by both the central clock in theSCN and the circadian clocks present in the pineal gland (Abe et al.,2002; Fukuhara et al., 2005). In contrast to light up-regulated Clock anti-phasic to dark up-regulated Per1-like and Cry2 in the pineal gland of parrunder LD (Huang et al., 2010b), the brain transcriptional repressors andactivators peaked before and after onset of daylight in postsomlts, respect-ively. Although it is uncertain if clock gene RNA levels can be comparedbetween parr and postsmolts, the differences of expression under LDmay be due to developmental changes. The acrophases of brain Per1-like,Per2, and Cry2 occurred a few hours later than those of the pineal genesand brain Per2, and Clock peaked in the beginning of the light phase,suggesting that a set of genes that peaked during the day or later at nightmay be regulated by pathways that are indirectly activated by melatoninor perhaps by a “master clock” located within a SCN‐like structure in thebrain. Yet, in salmonids, pineal melatonin is directly regulated by lightbut not by a circadian clock (Iigo et al., 2007), and salmonids may haveevolve a decentralized system in the pineal gland, independently of the


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eyes (Migaud et al., 2007). There is to date no evidence for the presenceof a functional circadian clock either in the pineal gland or retina,although they express clock genes in salmonids. Thus, further studiesshould clarify the role of the clock genes in the growth and stress axisand their expression in the hypothalamus.

The expression of c/ebpα and c/ebpδ exhibited daily variations in theliver and pineal gland, respectively, consistent with observations of rhyth-mic mRNA expression of c/ebp in the eye of the mollusc (Hattar et al.,2002), c/ebpβ in murine epididymal adipose tissue (Bray & Young, 2007),and c/ebpδ in the hamster pineal gland (Maronde et al., 2007). It hasbeen shown that C/EBP is involved in the regulation of arylalkylamineN-acetyltransferase (AANAT) mRNA in response to cyclic adenosinemonophosphate (cAMP) activation via the cAMP-response element-CCAAT complex in the promoter region (Baler et al., 1997) and that arole of adrenergic/cAMP signal transduction in regulating rhythmic geneexpression has been studied in rat pineal function (Bailey et al., 2009).These reports will provide a foundation for studies of the function of thesalmon c/ebpδ in the pineal gland. In a report by Bozek et al. (2009), C/EBP is a suggestive candidate for circadian transcriptional regulators in alarge-scale analysis of the CCG promoters, and circadian clock genes havebeen identified as targets of C/EBP in mouse NIH 3T3 cells (Gery et al.,2005). Thus, the rhythmic expression of salmon c/ebpα, c/ebpδ, and Per1-like supports the idea of a link between C/EBP proteins and the clockgene system. In addition, we found that expression of liver pparα mRNAshowed a dual peak, one in the evening and the other at dawn, whosevalues were statistically significant different from other timepoint valuesby ANOVA, but not by 24-h waveform cosinor analysis. This may bebecause the variable we are measuring varies over time, but not in a circa-dian manner, as not all biological rhythms are circadian. Targets of liverPPARα are involved in cellular lipid catabolism (Yamazaki et al., 2002),and the daily variation in the expression of lipogenic and cholesterogenicgenes were found to be attenuated or abolished in PPARα-null mice(Patel et al., 2001). As described in mammals (Jump, 2004), Atlanticsalmon PPAR and C/EBP have been suggested to be fatty acid–regulatedtranscriptional factors that play important roles in lipid metabolism inhepatocytes, adipocytes, and myotubes (Huang et al., 2010a; Klevelandet al., 2006; Vegusdal et al., 2004). PPARα and C/EBPα may act as poss-ible mediators for the daily rhythms of lipid metabolism in the liver,although this requires further study. Different sets of clock-controlledgenes across tissues were observed in the study of cycling transcripts inmice (Panda et al., 2002; Storch et al., 2002), suggesting liver c/ebpα,pparα, and pineal c/ebpδare putative clock-controlled genes in Atlanticsalmon. PPARs are expressed in the rat brain, of which the specificlocation, degree of expression, and physiological roles vary among the


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receptors (Ramanan et al., 2010). Consequently, it is important to furtherelucidate the role of individual nuclear transcription factors when inter-acting with tissue-specific ligands to perform to their own functioncoupled with clock gene expression in the pineal gland and liver.

The expression of C/EBP, PPAR, and clock genes did not show sig-nificant daily variations in the studied tissues under LL, with the excep-tion of pineal pdia3. The mRNA expression of pineal Clock, Per2, pparα,c/ebpα, and c/ebpδ increased under LL, whereas the liver Clock, Per2, andpparα expression decreased compared to under LD. Nonetheless, theincreasing or decreasing clock mRNAs were not observed in the brain,indicating a different role of clock genes across tissues. Although pinealPer2, c/ebpα, and c/ebpδ did not express at high levels in the studiedorgans during the day under LD, the increase of expression was observedunder LL in the pineal gland, but not in the liver. It is unknown whythere were opposite effects of LL on the expression of Per2, Clock, andpparα in the pineal gland and liver, and future studies should addressthis. These results will provide insight into clock gene system in salmonidsand provide considerable clues as to what is happening under LL con-ditions. Contrary to a lack of daily variations in the plasma levels of mela-tonin and cortisol and the expression clock genes, continuous light canenhance growth and muscle fiber recruitment (Johnston et al., 2003),affect mineralization, and delay osteoid incorporation in Atlantic salmonpostsmolts (Wargelius et al., 2009). Similar results have reported that con-tinuous light exposure affects the growth rate, timing of smoltification,and reproduction (Krakenes et al., 1991; Saunders & Henderson, 1988;Stefansson et al., 1991). Light and feeding effects on the daily rhythmshave been reported in clock genes of zebrafish (Per1 and Cry) by Sanchezand Sanchez-Vazquez (2009) and humoral daily rhythms of sea bream byLopez-Olmeda et al. (2009). In our experiments, LL-reared fish wereexposed to continuous light but 12 h cycles of feeding and starvationeach day and showed no variation in daily clock gene expression, indicat-ing that light has a stronger effect on clock gene expression than feedingin salmon. However, the connection between the earlier observed effectsof LL on growth and development and a disrupted rhythm in theexpression of clock genes imposed by LL as observed here remainselusive.

The most interesting effect of light was observed on pdia3 expression.The pdia3 mRNA appears to be rhythmically expressed in the pinealgland under both LD and LL, but the acrophases differed. Multiple func-tions of PDIA3 are known, i.e., to catalyze the oxidation, reduction, andisomerization of intra- and intermolecular disulfide bond formation in ER(Khanal & Nemere, 2007). We previously demonstrated that liver-expressed pdia3 and c/ebpδ may be important biomarkers for oxidant- andtoxicant-induced stress in smolts (Huang et al., 2009b). Although


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arrhythmic in the liver, the daily expression of pdia3 and c/ebpδ was onlypresent in the pineal gland under LD. Although it is unknown whetherthe significance of pineal pdia3 and c/ebpδ transcripts is related to physio-logical function, their expression may be coupled with clock geneexpression under LD in Atlantic salmon. Whether the observation thatpdia3 expression is not correlated with the arrhythmic expression of clockgenes under LL is related to the idea that an endogenous growth rhythmexists in Atlantic salmon under LL (Oppedal et al., 2006) remainsunknown. This connection and the importance of the rhythmic expressionof pineal pdia3 under both LD and LL need to be studied in more detail.

The present study indicates that light exerts an influence on the dailyvariation and overall expression of clock and nuclear transcription factorgenes in the pineal gland, brain, and liver of Atlantic salmon postsmolts,as well as levels of melatonin and cortisol. It is possible that light-dependent variations of expression reported here reflect interplaybetween daily and annual regulation. These changes can promote newapproaches for achieving a better understanding of how the clock genesystem is associated with the environmental, physiological, and geneticinteractions that occur in lifecycle transitions and their signaling pathwaybetween the brain and periphery. On the other hand, the integration ofcontinuous light for the timing of events, such as smoltification andsexual maturation, and its effect on the seasonal growth pattern, need tobe studied in more detail.

ACKNOWLEDGMENTS

We wish to thank Eva Mykkeltvedt, Jacob Wessels, and LeiknyFjeldstad for their excellent analytical work at NIFES and Dr. BenteE. Torstensen for reading through the manuscript. This work was sup-ported by the NIFES Internal Clock.

Declaration of Interest: The authors report no conflicts of interest.The authors alone are responsible for the content and writing of thepaper.

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